We have isolated the saxitoxin/tetrodotoxin binding component of the voltage-dependent sodium channel (SBC) from rat sarcolemma and have characterized some of its physical properties. We propose to extend our studies of this purified sodium channel protein in three general areas. First we will study the subunit composition of the purified channel protein. Bifunctional reagents will be used to cross-link subunits of the purified channel protein; spatially related subunits will be identified and their organization into higher MW complexes studied. Glycoprotein characteristics of the purified SBC subunits will be pursued using iodinated lectins and proteins separated on SDS-PAGE gradient gels. Binding of monoclonal antibodies raised to the purified channel will be studied to the protein subunits following electrophoretic separation and transfer to diazobenzyloxymethyl paper. Finally the STX/TTX binding site on the channel protein will be covalently labelled using a photoaffinity derivative of TTX, and the site identified on the subunits of the purified channel protein. Second, we will pursue reconstitution studies with the purified channel protein to access its capacity to gate ion movement. Initial studies with solubilized sarcolemma will lead to reconstitution of purified channel protein inphospholipid vesicles and ultimately to studies in black lipid membranes. Flux of labelled cations will first be measured using classical radioisotope techniques, but rapid kinetic analysis using fluorescent indicators and stopped-flow methodology will eventually be used. Third, we will develop a library of monoclonal antibodies directed against the sodium channel protein. Antibodies specific for the channel protein will be detected with a two level screening technique. Antibodies will be used to probe the channel subunit structure, to develop an affinity colum for channel purification, and to provide a means for identifying the sodium channel protein in situ at the light and electron microscopic level.